Membrane proteins and hydrophobic compounds are notoriously difficult to handle and represent two of the major challenges for pharmaceutical or life-science research and applications: (i) rendering insoluble hydrophobic compounds or membrane proteins soluble in aqueous solutions and (ii) the administration of such hydrophobic matter as therapeutic and diagnostic agents.
Membrane proteins are encoded by approximately 30% of all ORF (Wallin and von Heijne, Protein Science 1998 April; 7 (4):1029-38) and represent an important class of drug targets since the majority of drugs, i.e. more than 60%, target in fact this class of proteins (Overington et al., Nature Reviews Drug Discovery 5, 993-996 (December 2006)). Membrane proteins play essential roles in many biological processes, such as signal transduction, transport of molecules and energy, recognition and cell-to-cell communication. Yet, membrane proteins are difficult to study due to their insolubility and tendency to aggregate when extracted from their natural lipid bilayer environment. In order to maintain the integrity of membrane proteins, an artificial hydrophobic environment is needed. Here, detergent micelles are most commonly employed which may, however, negatively impact on biocompatibility, can have adverse affects on membrane protein activity and may interfere with experimental conditions for assays.
Another major pharmacological challenge is represented by the administration and delivery of hydrophobic compounds and/or hydrophobic proteins as therapeutic or diagnostic agents. Due to the limited solubility of these hydrophobic agents, they are prone to aggregation, leading to locally highly concentrated drug particles that may cause high toxicity, unwanted immune responses and render the drug inactive (Allen and Cullis, SCIENCE, 303 (5665): 1818-1822, Mar. 19, 2004).
Therefore, applications that incorporate hydrophobic agents such as membrane proteins, drugs or diagnostic compounds into soluble particles are highly desired. Current methods that address these two challenges involve amongst others liposomes and reconstituted high-density lipoprotein (rHDL) particles (Chan and Boxer, Current Opinion in chemical Biology 11:1-7, 2007).
EP 1 596 828 B1 describes disc-shaped bioactive agent delivery particles comprising an apolipoprotein which tightly surrounds a lipid bilayer in a double belt-like fashion. The interior of said particles is formed by the hydrophobic region of the lipid bilayer. This is in contrast to liposomes, which are closed spherical bilayer shells containing an aqueous interior. The disc-shaped bioactive agent delivery particles described in EP 1 596 828 B1 have a Stokes diameter of about 10 nm and are proposed for use as delivery vehicles for hydrophobic pharmaceutical drugs such as amphotericin B or camptothecin.
EP 1 345 959 B1 describes a similar type of nanoscale particle with a diameter of about 10 nm and a height of about 5.5 nm. The particles are disc-shaped and composed of (i) an artificial membrane scaffold protein, (ii) a phospholipid bilayer and (iii) at least one hydrophobic or partially hydrophobic membrane protein. Said membrane scaffold protein again surrounds the lipid bilayer in a double belt-like fashion and is a derivative or a truncated form of human apolipoprotein A-1, lacks the N-terminal globular domain of human apolipoprotein A-1, is amphipathic and forms at least one α-helix and will, in aqueous environment, self-assemble with a phospholipid or mixture of phospholipids into a nanoscale particle of this discoidal shape. Such an engineered membrane scaffold protein (MSP) shall provide stability, size homogeneity and useful functionalities to the nanoscale discoidal lipoprotein particle.
However, there are several drawbacks with this currently available nanodisc technology in that, for example, a removal of detergent is required during assembly of the particles. Moreover, the size homogeneity provided by the tight double-belt like fit of the apolipoprotein-derived MSP seems to go at the expense of a fixed minimum particle size and a limitation as to the maximum diameters obtainable with the methods of the prior art.
The saposin-family comprises 4 small (˜80 amino acids) proteins, saposin A to D, that bind and/or interact with lipids and function as essential cofactors for several lysosomal enzymes in sphingolipid catabolism (cf. Bruhn, Biochem J. (2005) 389, 249-257 and references cited therein). Saposins have been described to prefer negatively charged lipids and low pH, exhibiting markedly increased activities at acidic pH, with a pH optimum at the intra-lysosomal pH of 4.75. Saposin A, B, C, and D are proteolytically hydrolyzed from a single large precursor protein, prosaposin. The complete amino acid sequences for saposins A, B, C and D have been reported as well as the genomic organization and cDNA sequence of prosaposin (O'Brien et al. (1988) Science 241, 1098-1101; Furst et al (1992) Biochim Biophys Acta 1126: 1-16).
Saposin C is capable of inducing membrane fusion of phospholipid-containing vesicles in an acidic environment (Archives of Biochemistry and Biophysics 2003 Jul. 1; 415(1): 43-53), a feature not exhibited by the other saposins. Qi et al. (2009) Clin Cancer Res 15(18):5840-5851 report on saposin C-coupled dioleoylphosphatidylserine nanovesicles (SapC-DOPS) that contain an aqueous interior, have a mean diameter of about 190 nm and show tumor-targeting activity in vivo. In SapC-DOPS, saposin C or a peptide derived thereof acts as homing peptide for the liposome it is attached to. Saposin C then targets the liposome to cancer cells exposing phosphatidylserine on the outer leaflet of the cell membrane. The authors believe that a unique acidic microenvironment around cancer cells due to extracellular leakage of lysosomal enzymes makes tumor tissue an optimal target for saposin C. According to Qi et al., SapC-DOPS liposomes are prepared by drying solvent-dissolved phospholipids under N2 (g), dispersing the dry phospholipids in acidic buffer (pH 5) containing purified saposin C, diluting the mixture 50× in a physiologic aqueous solution and facilitating nanovesicle assembly by subsequent sonication.
Popovic et al., PNAS, Vol. 109, No. 8 (2012) 2908-2912 report on the structure of saposin A detergent discs. Saposin A exists in a soluble and a lipid/detergent-bound state. In the absence of lipid, saposin A adopts a closed monomeric apo conformation. By contrast, the saposin A detergent disc structure reported by Popovic et al. reveals two chains of saposin A in an open conformation encapsulating 40 internally bound detergent molecules organized in a highly ordered bilayer-like hydrophobic core.
Besides the crystallization of saposin A detergent discs, Popovic et al. also describe the preparation of soluble lipid-saposin A complexes at pH 4.75 by a method requiring multiple steps. First, a uniform fraction of large unilamellar liposome vesicles is prepared by drying chloroform-dissolved lipids under N2 (g), dispersing the dry lipids by vortex mixing in acidic buffer (50 mM sodium acetate pH 4.8, 150 mM NaCl), submitting the suspension to 10 cycles of freezing and thawing, blending in a vortex mixer for 5 min and extruding the mixture through a 200 nm filter. Mixing the thus prepared large unilamellar liposome vesicles with purified saposin A in acidic buffer resulted in soluble lipid-saposin A particles. The particle showed a narrow size distribution around an average hydrodynamic (Stokes) radius of 3.2 nm and contained about 5:1 lipid molecules per saposin A chain. The exact size of the particles was only moderately affected by the lipid to protein molar ratio and the composition of the liposomes. The authors observed similar 3.2 nm particles regardless of whether or not anionic phospholipids, cholesterol, or glycosphingolipids were present in the liposomal mixtures. In all cases, a single peak was observed in the size range of a Stokes radius of 3.2 nm, indicating a relatively narrow distribution of species. Hence, the technology of this publication is limited to a pH value of 4.75, to the aforementioned size of the particles, and includes a laborious upstream liposome preparation step.
Against this background, there is a need for a reliable and easy to perform method for generating stable, defined lipoprotein-particle compositions for the solubilization of membrane proteins and other hydrophobic compounds. This is particularly true in view of the elaborate and multi-step processes disclosed in the prior art for preparing discoidal lipoprotein particles.
A wide variety of hydrophobic agents could potentially benefit from the apolipoprotein- or saposin A-derived nanodisc technology described in the prior art. However, due to the 3.2 nm size limitation of the saposin A derived particles, it seems that—if at all—only small molecules may be incorporated into such particles at the acidic pH disclosed therein. Whereas bulky hydrophobic compounds and large biomolecules such as (oligomeric) membrane proteins can be incorporated into the Apolipoprotein A derived nanodiscs of the prior art, the maximum possible diameter is still limited by the double-belt like Apolipoprotein A perimeter of these particles. In addition, the 10 nm Apolipoprotein A derived nanodiscs may be too large for certain applications. Hence, there is a need for advanced nanodisc technology and superior liproprotein particles with flexible and controllable size ranges featuring the ability to adapt to the respective size of the hydrophobic agent that is to be incorporated into the lipoprotein particles. Such particles shall allow for simple integration of membrane proteins and other hydrophobic components which, for example, can be pharmaceutically or biologically active compounds or diagnostic compounds.